Bilin pigments, when associated with proteins, exhibit a wide variety of photophysical properties, i.e. intense fluorescence, photochemical interconversions, and radiationless de-excitation. Differences in the protonation state, conformation and/or ionic environment of bilin pigments can significantly alter their absorption properties. In this way, the protein moiety of biliproteins tunes the spectrum of their bilin chromophore. Plants, some bacteria, and fungi contain phytochromes—self-assembling biliproteins that act as light sensors to modulate growth and development (Frankenberg and Lagarias (2003) Pp. 211-235 In: The Porphyrin Handbook. Chlorophylls and Bilins: Biosynthesis Structure and Degradation., K. M. Kadish, K. M. Smith, and R. Guilard, Editors. Academic Press: New York; Lagarias and Lagarias (1989) Proc. Natl. Acad. Sci. USA, 86(15): 5778-5780). Phytochromes' covalently bound bilin prosthetic groups photoisomerize upon absorption of light enabling the protein to photointerconvert between two distinct species, which have absorption maxima in the red and NIR region. Unlike the intensely fluorescent phycobiliproteins, components of the photosynthetic antennae of algae, native phytochromes are non-fluorescent biliproteins because this photoconversion process is so efficient.
The optical properties of phytochromes are highly malleable, as shown by the spectral diversity of phytochromes in nature. In plants, algae, and cyanobacteria, phytochromes are associated with the linear tetrapyrroles phytochromobilin (PφB) or phycocyanobilin (PCB). Binding of an apophytochrome to the unnatural bilin precursor, phycoerythrobilin (PEB) however, affords a strongly fluorescent phytochrome known as a phytofluor, that is unable to isomerize upon light absorption (Murphy and Lagarias (1997) Current Biology, 7: 870-876). Phytofluors have been shown to be useful probes in living cells, however, addition of exogenous unnatural bilin precursors is generally necessary. Recently, a new class of phytochromes from bacteria and fungi was identified that attach a different bilin chromophore, biliverdin (BV), to an apparently distinct region of the apoprotein (Lamparter et al. (2002) Proc. Natl. Acad. Sci. USA, 99(18): 11628-11633). These studies indicate that molecular evolution has occurred in nature to produce phytochrome mutants with novel spectroscopic properties.
The jellyfish green fluorescent protein (GFP) has revolutionized cell biological studies, allowing for the visualization of protein dynamics in real-time within living cells by in frame fusion to a gene of interest. Applications of GFP and related fluorescent proteins of the GFP family include investigation of protein-protein interactions, spatial and temporal gene expression, and sub-cellular localization. GFPs have also been used to label organelles, to image pH and calcium fluxes, and to test targeting peptides (Chiesa et al. (2001) Biochemical J., 355(Part 1): 1-12).
Despite their utility, as with any technology, GFPs have inherent limitations. During the maturation of the GFP chromophore, where three adjacent amino acid residues within the protein cyclize (S65, Y66, G67) and the tyrosine residue is thereafter dehydrogenated, cytotoxic hydrogen peroxide is produced (Cubitt et al. (1995) Trends In Biochemical Sciences, 20(11): 448-455). GFPs are typically homodimers, a property that can interfere with the native function of the fused protein of interest. GFPs are also temperature and pH-sensitive and can be highly susceptible to photobleaching and oxidation. While some of these problems have been overcome by directed evolution (Zhang et al. (2002) Nature Reviews Molecular Cell Biology, 3(12): 906-918), many cannot. In particular, directed evolution has been used to engineer an array of color variants of GFP ranging from blue, to cyan, to yellow (Heim and Tsien (1996) Current Biology, 6: 178-182). Despite the multiple engineering efforts to date, long wavelength red and NIR emitting GFP variants have not yet been isolated. The recent identification of the orange fluorescent protein DsRed (excitation maximum 558 nm, and emission maximum 583 nm) from the coral genus Discosoma has sparked a great deal of interest due to its longer wavelength emission properties (Matz et al. 91999) Nature Biotechnology, 17(10): 969-973). However, DsRed exhibits a tendency to tetramerize and long incubation periods are needed to reach steady-state fluorescence levels (Zhang et al. (2002) Nature Reviews Molecular Cell Biology, 3(12): 906-918).